Bioactive Chaetoglobosins from the Mangrove Endophytic Fungus Penicillium chrysogenum

A novel chaetoglobosin named penochalasin I (1) with a unprecedented six-cyclic 6/5/6/5/6/13 fused ring system, and another new chaetoglobosin named penochalasin J (2), along with chaetoglobosins G, F, C, A, E, armochaetoglobosin I, and cytoglobosin C (3–9) were isolated from the culture of Penicillium chrysogenum V11. Their structures were elucidated by 1D, 2D NMR spectroscopic analysis and high resolution mass spectroscopic data. The absolute configuration of compounds 1 and 2 were determined by comparing the theoretical electronic circular dichroism (ECD) calculation with the experimental CD. Compound 1 was the first example, with a six-cyclic fused ring system formed by the connection of C-5 and C-2′ of the chaetoglobosin class. Compounds 5–8 remarkably inhibited the plant pathogenic fungus R. solani (minimum inhibitory concentrations (MICs) = 11.79–23.66 μM), and compounds 2, 6, and 7 greatly inhibited C. gloeosporioides (MICs = 23.58–47.35 μM), showing an antifungal activity higher than that of carbendazim. Compound 1 exhibited marked cytotoxicity against MDA-MB-435 and SGC-7901 cells (IC50 < 10 μM), and compounds 6 and 9 showed potent cytotoxicity against SGC-7901 and A549 cells (IC50 < 10 μM).


Chemical Structure Elucidation
Penochalasin I (1) was obtained as yellow crystal. Its molecular formula was established as C32H34N2O4 by HRESIMS (high resolution electrospray ionization mass spectroscopy) at m/z 511.2614 ([M + H] + , cacld. for C32H35N2O4, 511.2591), indicating 17 degrees of unsaturation. The 13 C NMR and DEPT (distortionless enhancement by polarization transfer) spectra of compound 1 (Table 1) displayed 32 carbons, including four methyls, two sp 3 hybridized methylenes, five sp 3 and ten sp 2 hybridized methines, and six sp 2 hybridized quaternary, an amide (δC 173.9), and two ketone (δC 201.7 and 198.2) carbons. The 1 H NMR spectrum of 1 (Table 1)  , and an amide proton (δH 7.61, exchangeable). The 13 C NMR data combined with the characteristic 1 H NMR signals suggested that compound 1 was most likely a chaetoglobosin-based alkaloid with close resemblance to chaetoglobosin J [6]. Based on the evidence above, the main differences between compounds 1 and chaetoglobosin J were as follows: (1) The secondary 11-methyl (δH 1.42, d) in chaetoglobosin J had been replaced by a tertiary methyl (δH 1.41, s) in 1; (2) The indolyl group in 1

Chemical Structure Elucidation
Penochalasin I (1) was obtained as yellow crystal. Its molecular formula was established as C 32  , and an amide proton (δ H 7.61, exchangeable). The 13 C NMR data combined with the characteristic 1 H NMR signals suggested that compound 1 was most likely a chaetoglobosin-based alkaloid with close resemblance to chaetoglobosin J [6]. Based on the evidence above, the main differences between compounds 1 and chaetoglobosin J were as follows: (1) The secondary 11-methyl (δ H 1.42, d) in chaetoglobosin J had been replaced by a tertiary methyl (δ H 1.41, s) in 1; (2) The indolyl group in 1 had a 2,3-substituted pattern instead of the 3-substituted pattern (chaetoglobosin J). Moreover, a subsequent comparison between the 13 C NMR spectra and degrees of unsaturation revealed by the molecular formulas of 1 and chaetoglobosin J showed that 1 had one more cyclic ring than the latter. These results suggested the connectivity of C-5 and C-2 of 1, which established an unprecedented 6/5/6/5/6/13 six cyclic system. This deduction was supported by HMBC (heteronuclear multiple bond correlation) correlations of H-11 to C-2 , C-4, and C-5, and of H-12 to C-5, C-6, and C-7. Finally, comprehensive HMBC and 1H-1H COSY (chemical-shift correlation spectroscopy) analysis ( Figure 2) allowed the entire assignment of the proton and carbon signals for 1. To the best to our knowledge, compound 1 featured a unique carbon skeleton with a six-cyclic 6/5/6/5/6/13 fused ring system, and was the first member of the chaetoglobosin family with an unprecedented six-membered ring formed by the connection of C-5 and C-2 .        Penochalasin J (2) (Table 1) and HSQC (heteronuclear singular quantum correlation) spectroscopic data for 2 revealed 32 carbon signals, including three carbonyl groups (δC 210.2, 209.7.5, and 175.0), 14 olefinic and aromatic carbons, an sp 3 quaternary carbon, five sp 3 methylene groups, five sp 3 methines, and four methyl groups. The protons and protonated carbon resonances in the NMR spectra of 2 were unambiguously assigned by 1 H-1 H COSY and HSQC experiments. The general features of its NMR data closely resembled those of penochalasin G, a cytochalasan-based alkaloid characterized from an endophytic strain of Penicillium sp. OUPS-19, isolated from the marine alga Enteromorpha intestinalis [10]. The only significant difference between the two compounds was the absence of a hydroxyl in 2 at C-19. The observed HMBC correlations ( Figure 2) from H-19a and H-19b to C-17, C-18, C-18′, C-20, and C-21 confirmed the above deduction.
The relative configuration of 2 was determined by analyzing its NOESY correlations (Figure 3) of those protons. NOE correlations between H-5 and H-8, and no observable NOE correlations between H-4 and H-8 implied that the cyclohexane ring was in a twist-boat conformation, and both H-5 and H-8 were assigned as β-orientation. 10 (Table 1) and HSQC (heteronuclear singular quantum correlation) spectroscopic data for 2 revealed 32 carbon signals, including three carbonyl groups (δ C 210.2, 209.7.5, and 175.0), 14 olefinic and aromatic carbons, an sp 3 quaternary carbon, five sp 3 methylene groups, five sp 3 methines, and four methyl groups. The protons and protonated carbon resonances in the NMR spectra of 2 were unambiguously assigned by 1 H-1 H COSY and HSQC experiments. The general features of its NMR data closely resembled those of penochalasin G, a cytochalasan-based alkaloid characterized from an endophytic strain of Penicillium sp. OUPS-19, isolated from the marine alga Enteromorpha intestinalis [10]. The only significant difference between the two compounds was the absence of a hydroxyl in 2 at C-19. The observed HMBC correlations ( Figure 2) from H-19a and H-19b to C-17, C-18, C-18 , C-20, and C-21 confirmed the above deduction.
More detailed spectra and ECD computational details of new compounds are available in the Supplementary Materials (Figures S1-S24 and Tables S1-S5).
More detailed spectra and ECD computational details of new compounds are available in the Supplementary Materials (Figures S1-S24 and Tables S1-S5).
To our best knowledge, this is the first report of antifungal activities against the test plant pathogenic fungi of the seven isolated chaetoglobosins. Compounds 1-9 were examined for their cytotoxic activities against three human tumor cell lines, including a human breast cancer cell line (MDA-MB-435), a human gastric cancer cell line (SGC-7901), and a human lung adenocarcinoma epithelial cell line (A549) by MTT (3-(4,5dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide) assay, as described in the liturature [24] using epirubicin as positive control. The results are presented in Table 3. As shown in Table 3, Penochalasin I (1) exhibited marked cytotoxic activities against MDA-MB-435 and SGC-7901 cell lines (IC 50 < 10 µM). Two other compounds, chaetoglobosin A (6) and cytoglobosin C (9) showed potent cytotoxicity against both SGC-7901 and A549 cell lines (IC 50 (half maximal inhibitory concentration) < 10 µM). In contrast, armochaetoglobin I (8) showed no inhibitory activities against any of the test cell lines (IC 50 > 40 µM), and chaetoglobosin E (7) was inactive towards both MDA-MB-435 and SGC-7901 cell lines (IC 50 > 40 µM). The remaining compounds exhibited moderate cytotoxicity, with IC 50 values ranging from 12.58 to 38.77 µM. Our results were consistent with the structure-activity relationships reported in previous studies [19]. Compound 5 exhibited higher cytotoxic activities against the test cell lines than compound 3, suggesting that the epoxide ring at C-6/C-7 favoured an improvement of cytotoxicity. Compound 4 showed lower cytotoxic activities than compound 5, indicating that the reduction of 20-carbonyl into 20-hydroxyl could decrease the cytotoxicity. This deduction could also be concluded by comparison of the cytotoxic activities between compounds 3 and 7. In previous investigations, the known chaetoglobosins covered in the study had shown cytotoxicity against many different human cancer cell lines [15,16,[27][28][29]32,33]. To our best knowledge, there were no previous reports on the cytotoxicity of these chaetoglobosins to MDA-MB-435, A549 and SGC-7901 cell lines, except compounds 8 and 9, which were tested on the A549 cell line [28,29] with IC 50 values almost identical to our current results.

General Experimental Procedures
NMR experiments were carried out on a Bruker AVIII 600 MHz NMR spectrometer (Bruker BioSpin GmbH company, Rheinstetten, Germany) ( 1 H 600 MHz, 13 C 150 MHz), with tetramethylsilane as the internal standard. Optical rotations were recorded with an MCP 300 (Anton Paar, Shanghai, China) polarimeter at 28 • C. UV spectra were measured on a PERSEE TU-1900 spectrophotometer. IR spectra were carried out on a Nicolet Nexus 670 spectrophotometer, in KBr discs. CD spectra were measured on a Chirascan™ CD spectrometer (Applied Photophysics, London, UK). ESIMS spectra were recorded on a Finnigan LCQ-DECA mass spectrometer, and HRESIMS spectra were recorded on a Thermo Fisher Scientific Q-TOF mass spectrometer. Column chromatography (CC) was performed on silica gel (200-300 mesh, Qingdao Marine Chemical Factory, Qingdao, China) and Sephadex LH-20 (Amersham Pharmacia Biotech., Uppsala, Sweden). Thin-layer chromatography (TLC) was performed on silica gel plates (Qingdao Huang Hai Chemical Group Co., Qingdao, China, G60, F-254). The high-performance liquid chromatography (HPLC) separation was performed on a Varian Prostar 210 system equipped with a Prostar 320 UV detector on a preparative Hypersil C-18 BDS column (250 × 21.2 mm, L × ID, 5 µm Varian Dynamax, Thermo Fisher Scientific Inc., Waltham, MA, USA). All other chemicals used were analytical grade.

Fungal Material
The strain of Penicillium chrysogenum V11 was isolated from the vein of Myoporum bontioides A. Gray in Leizhou Peninsula and deposited in the College of Materials and Energy, South China Agriculture University, Guangdong Province, China. The fungus was identified using a molecular biological protocol by DNA amplification and sequencing of the ITS region. A BLAST search result showed that the sequence was the most similar (99%) to the sequence of Penicillium chrysogenum (compared to KR011761.1, KX349473.1, JQ015265.1, and KF039676.1). The sequence data obtained from the fungal strain was submitted to GenBank with accession number KX777253.

Antifungal Activity Assay
The following four phytopathogenic fungi were used for bioassay: Colletotrichum musae (Berk. and M. A. Curtis) Arx. (C. musae), Colletotrichum gloeosporioides (Penz) Sacc. (C. gloeosporioides), Penicillium italicum Wehme (P. italicm), Rhizoctonia solani Kühn (R. solani). They were obtained from the College of Agriculture, South China Agricultural University. The antimicrobial activities of the pure compounds against the four phytopathogenic fungi were determined by the broth dilution method as described in the previous literature to get the minimum inhibitory concentration (MIC) [26]. Carbendazim and the solvent were used as positive and negative control, respectively.

Cytotoxicity Assay
Cytotoxicity activities of the compounds against three human tumor cell lines, including a human breast cancer cell line (MDA-MB-435), a human gastric cancer cell line (SGC-7901), and a human lung adenocarcinoma epithelial cell line (A549) were tested by MTT method, as described in a previous report [25]. Epirubicin was used as the positive control.

Conclusions
This study led to the isolation of two new chaetoglobosins-penochalasins I (1) and J (2)-which enriched the library of natural compounds. Notably, penochalasin I (1) possessed a unique six-cyclic 6/5/6/5/6/13 fused ring structure and was the first example with such a system formed by the connection of C-5 and C-2 of the chaetoglobosin class. Compounds 5-8 exhibited potent antifungal activities against the plant pathogen R. solani (MICs = 23.66, 11.83, 11.79, and 12.11 µM), and compounds 2, 6, and 7 displayed potent antifungal activities towards C. gloeosporioides (MICs = 25.08, 47.35, and 23.58 µM), with results that were better than the positive control, carbendazim. Compound 1 exhibited marked cytotoxic activities against MDA-MB-435 and SGC-7901 cell lines (IC 50 < 10 µM), while compounds 6 and 9 showed potent cytotoxicity against both SGC-7901 and A549 cell lines (IC 50 < 10 µM). These bioactive penochalasins in the endophytic fungus might play a defensive role by inhibiting other invasive fungi, providing more nutrition and living space. They could be worthy of consideration for the development and research of antifungal and/or antitumor agents.